Many organs such as the mammalian liver possess intrinsic information about their final size, yet the molecular mechanisms that govern organ size in animal development and regeneration remain poorly understood. The recent discovery of the Hippo signaling pathway provides an unprecedented entry point to resolving this long- standing puzzle in biology. First discovered in Drosophila, the Hippo signaling pathway is an evolutionarily conserved regulator of organ size. Central to the Hippo pathway is a kinase cascade leading from the protein kinase Hpo/Mst to a transcription factor complex formed between coactivator Yki/YAP and DNA-binding transcription factor Sd/TEAD. Consistent with the critical role of Hippo signaling in normal tissue homeostasis, the YAP protein is overexpressed or hyperactivated in a wide spectrum of human cancers due to YAP locus amplification or genetic/epigenetic inactivation of upstream tumor suppressors. Small molecule inhibitors of YAP will not only provide important tools for pharmacological manipulation of Hippo signaling, but also bear tremendous potential for developing therapeutic drugs against human diseases caused by defective Hippo signaling. To this end, we have designed and functionally validated a primary high-throughput screen assay to discover chemicals capable of inhibiting the interaction of coactivator Yki/YAP and its cognate transcription factor Sd/TEAD. We have collaborated the Chemical Genomics Branch at NIH Chemical Genomics Center (NCGC) to miniaturize this assay to 1536-plate format and successfully conducted a pilot screen with the Library of Pharmacologically Active Compounds 1280 (LOPAC1280). In this proposed research, we aim to first implement this target-based FRET assay in a high-throughput screen against the National Institute of Health- Molecular Libraries Small Molecule Repository (NIH-MLSMR) collection covering a wide range of chemical space at the NCGC screening center. Next, we will validate the primary screen hits using a plethora of pre- established follow-up assays to remove false positives and prioritize the hit set. Finally we will test the hits in two well-characterized Hippo pathway-specific mouse models of YAP-induced liver hyperplasia and liver cancer. Taken together, our proposed research represents the first-of-its-kind large-scale screening campaign to discover lead compounds targeting the Hippo signaling pathway.